Ebola and Marburg viruses belong to the family Filoviridae and can cause fatal hemorrhagic fevers characterized by widespread tissue destruction. Because of the safety concerns, these viruses are designated as the bio-safety level 4 agents. Progress in elucidation of the mechanisms of Ebola entry and pathogenesis has been hampered partly due to the safety concern. However, various surrogate systems based on the viral pseudotyping technique have been established to investigate the entry mechanism of Ebola viruses. In this application, we demonstrate that Ebola glycoprotein (GP) can be efficiently incorporated onto HIV viral particles, and the pseudotyped virions are highly infectious, as reported by others. More importantly, we show that we can use this system as a powerful entry assay to elucidate the entry mechanism of Ebola viruses. Our preliminary results clearly illustrate how we can use this pseudotyped system to dissect the roles of both subunits (GP1 and GP2) of Ebola GP in receptor recognition and viral entry. This application will focus on the following specific aims: (1) Characterization of the roles of the charged residues of the GP2 hairpin in Ebola entry. (2) Analysis of the receptor binding region of GP1 in Ebola entry. (3) Identification and characterization of the potential Ebola receptor(s). These studies will provide important information regarding how Ebola GP interacts with the cellular receptor(s) and how Ebola GP mediates efficient membrane fusion and viral entry. Furthermore, elucidation of the roles of GP1 and GP2 in Ebola entry will help us design effective Ebola entry inhibitors which may provide therapeutic treatments against Ebola infection and disease. Therefore, the research work proposed in this application is not only important for elucidating the basic entry mechanism of Ebola viruses, but also important for developing potentially powerful treatments to combat this deadly pathogen.